Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Apr;178(7):2086–2093. doi: 10.1128/jb.178.7.2086-2093.1996

Characterization of the stable maintenance properties of the par region of broad-host-range plasmid RK2.

P A Sobecky 1, C L Easter 1, P D Bear 1, D R Helinski 1
PMCID: PMC177909  PMID: 8606188

Abstract

A 3.2-kb fragment encoding five genes, parCBA/DE, in two divergently transcribed operons promotes stable maintenance of the replicon of the broad-host-range plasmid RK2 in a vector-independent manner in Escherichia coli. The parDE operon has been shown to contribute to stabilization through the postsegregational killing of plasmid-free daughter cells, while the parCBA operon encodes a resolvase, ParA, that mediates the resolution of plasmid multimers through site-specific recombination. To date, evidence indicates that multimer resolution alone does not play a significant role in RK2 stable maintenance by the parCBA operon in E. coli. It has been proposed, instead, that the parCBA region encodes an additional stability mechanism, a partition system, that ensures that each daughter cell receives a plasmid copy at cell division. However, studies carried out to date have not directly determined the plasmid stabilization activity of the parCBA operon alone. An assessment was made of the relative contributions of postsegregational killing (parDE) and the putative partitioning system (parCBA) to the stabilization of mini-RK2 replicons in E. coli. Mini-RK2 replicons carrying either the entire 3.2-kb (parCBA/DE) fragment or the 2.3-kb parCBA region alone were found to be stably maintained in two E. coli strains tested. The stabilization found is not due to resolution of multimers. The stabilizing effectiveness of parCBA was substantially reduced when the plasmid copy number was lowered, as in the case of E. coli cells carrying a temperature-sensitive mini-RK2 replicon grown at a nonpermissive temperature. The presence of the entire 3.2-kb region effectively stabilized the replicon, however, under both low- and high-copy-number-conditions. In those instances of decreased plasmid copy number, the postsegregational killing activity, encoded by parDE, either as part of the 3.2-kb fragment or alone played the major role in the stabilization of mini-RK2 replicons within the growing bacterial population. Our findings indicate that the parCBA operon functions to stabilize by a mechanism other than cell killing and resolution of plasmid multimers, while the parDE operon functions solely to stabilize plasmids by cell killing. The relative contribution of each system to stabilization depends on plasmid copy number and the particular E. coli host.

Full Text

The Full Text of this article is available as a PDF (265.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abeles A. L., Friedman S. A., Austin S. J. Partition of unit-copy miniplasmids to daughter cells. III. The DNA sequence and functional organization of the P1 partition region. J Mol Biol. 1985 Sep 20;185(2):261–272. doi: 10.1016/0022-2836(85)90402-4. [DOI] [PubMed] [Google Scholar]
  2. Austin S., Ziese M., Sternberg N. A novel role for site-specific recombination in maintenance of bacterial replicons. Cell. 1981 Sep;25(3):729–736. doi: 10.1016/0092-8674(81)90180-x. [DOI] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blakely G., May G., McCulloch R., Arciszewska L. K., Burke M., Lovett S. T., Sherratt D. J. Two related recombinases are required for site-specific recombination at dif and cer in E. coli K12. Cell. 1993 Oct 22;75(2):351–361. doi: 10.1016/0092-8674(93)80076-q. [DOI] [PubMed] [Google Scholar]
  5. Bravo A., Ortega S., de Torrontegui G., Díaz R. Killing of Escherichia coli cells modulated by components of the stability system ParD of plasmid R1. Mol Gen Genet. 1988 Dec;215(1):146–151. doi: 10.1007/BF00331316. [DOI] [PubMed] [Google Scholar]
  6. Bravo A., de Torrontegui G., Díaz R. Identification of components of a new stability system of plasmid R1, ParD, that is close to the origin of replication of this plasmid. Mol Gen Genet. 1987 Nov;210(1):101–110. doi: 10.1007/BF00337764. [DOI] [PubMed] [Google Scholar]
  7. Bron S., Bosma P., van Belkum M., Luxen E. Stability function in the Bacillus subtilis plasmid pTA 1060. Plasmid. 1987 Jul;18(1):8–15. doi: 10.1016/0147-619x(87)90073-4. [DOI] [PubMed] [Google Scholar]
  8. Burkardt H. J., Riess G., Pühler A. Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical. J Gen Microbiol. 1979 Oct;114(2):341–348. doi: 10.1099/00221287-114-2-341. [DOI] [PubMed] [Google Scholar]
  9. Carter P., Bedouelle H., Winter G. Improved oligonucleotide site-directed mutagenesis using M13 vectors. Nucleic Acids Res. 1985 Jun 25;13(12):4431–4443. doi: 10.1093/nar/13.12.4431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Davis T. L., Helinski D. R., Roberts R. C. Transcription and autoregulation of the stabilizing functions of broad-host-range plasmid RK2 in Escherichia coli, Agrobacterium tumefaciens and Pseudomonas aeruginosa. Mol Microbiol. 1992 Jul;6(14):1981–1994. doi: 10.1111/j.1365-2958.1992.tb01371.x. [DOI] [PubMed] [Google Scholar]
  11. Dodd H. M., Bennett P. M. Location of the site-specific recombination system of R46: a function necessary for plasmid maintenance. J Gen Microbiol. 1986 Apr;132(4):1009–1020. doi: 10.1099/00221287-132-4-1009. [DOI] [PubMed] [Google Scholar]
  12. Eberl L., Givskov M., Schwab H. The divergent promoters mediating transcription of the par locus of plasmid RP4 are subject to autoregulation. Mol Microbiol. 1992 Jul;6(14):1969–1979. doi: 10.1111/j.1365-2958.1992.tb01370.x. [DOI] [PubMed] [Google Scholar]
  13. Eberl L., Kristensen C. S., Givskov M., Grohmann E., Gerlitz M., Schwab H. Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4. Mol Microbiol. 1994 Apr;12(1):131–141. doi: 10.1111/j.1365-2958.1994.tb01002.x. [DOI] [PubMed] [Google Scholar]
  14. Funnell B. E. Participation of Escherichia coli integration host factor in the P1 plasmid partition system. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6657–6661. doi: 10.1073/pnas.85.18.6657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gallie D. R., Kado C. I. Agrobacterium tumefaciens pTAR parA promoter region involved in autoregulation, incompatibility and plasmid partitioning. J Mol Biol. 1987 Feb 5;193(3):465–478. doi: 10.1016/0022-2836(87)90260-9. [DOI] [PubMed] [Google Scholar]
  16. Gerdes K., Rasmussen P. B., Molin S. Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells. Proc Natl Acad Sci U S A. 1986 May;83(10):3116–3120. doi: 10.1073/pnas.83.10.3116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gerdes K., Thisted T., Martinussen J. Mechanism of post-segregational killing by the hok/sok system of plasmid R1: sok antisense RNA regulates formation of a hok mRNA species correlated with killing of plasmid-free cells. Mol Microbiol. 1990 Nov;4(11):1807–1818. doi: 10.1111/j.1365-2958.1990.tb02029.x. [DOI] [PubMed] [Google Scholar]
  18. Gerlitz M., Hrabak O., Schwab H. Partitioning of broad-host-range plasmid RP4 is a complex system involving site-specific recombination. J Bacteriol. 1990 Nov;172(11):6194–6203. doi: 10.1128/jb.172.11.6194-6203.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Haugan K., Karunakaran P., Blatny J. M., Valla S. The phenotypes of temperature-sensitive mini-RK2 replicons carrying mutations in the replication control gene trfA are suppressed nonspecifically by intragenic cop mutations. J Bacteriol. 1992 Nov;174(21):7026–7032. doi: 10.1128/jb.174.21.7026-7032.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Helsberg M., Eichenlaub R. Twelve 43-base-pair repeats map in a cis-acting region essential for partition of plasmid mini-F. J Bacteriol. 1986 Mar;165(3):1043–1045. doi: 10.1128/jb.165.3.1043-1045.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hwang D. S., Kornberg A. Opening of the replication origin of Escherichia coli by DnaA protein with protein HU or IHF. J Biol Chem. 1992 Nov 15;267(32):23083–23086. [PubMed] [Google Scholar]
  22. Jaffé A., Ogura T., Hiraga S. Effects of the ccd function of the F plasmid on bacterial growth. J Bacteriol. 1985 Sep;163(3):841–849. doi: 10.1128/jb.163.3.841-849.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jensen R. B., Dam M., Gerdes K. Partitioning of plasmid R1. The parA operon is autoregulated by ParR and its transcription is highly stimulated by a downstream activating element. J Mol Biol. 1994 Mar 11;236(5):1299–1309. doi: 10.1016/0022-2836(94)90059-0. [DOI] [PubMed] [Google Scholar]
  24. Jovanovic O. S., Ayres E. K., Figurski D. H. Host-inhibitory functions encoded by promiscuous plasmids. Transient arrest of Escherichia coli segregants that fail to inherit plasmid RK2. J Mol Biol. 1994 Mar 18;237(1):52–64. doi: 10.1006/jmbi.1994.1208. [DOI] [PubMed] [Google Scholar]
  25. Krause M., Guiney D. G. Identification of a multimer resolution system involved in stabilization of the Salmonella dublin virulence plasmid pSDL2. J Bacteriol. 1991 Sep;173(18):5754–5762. doi: 10.1128/jb.173.18.5754-5762.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kües U., Stahl U. Replication of plasmids in gram-negative bacteria. Microbiol Rev. 1989 Dec;53(4):491–516. doi: 10.1128/mr.53.4.491-516.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lane D., de Feyter R., Kennedy M., Phua S. H., Semon D. D protein of miniF plasmid acts as a repressor of transcription and as a site-specific resolvase. Nucleic Acids Res. 1986 Dec 22;14(24):9713–9728. [PMC free article] [PubMed] [Google Scholar]
  28. Lehnherr H., Yarmolinsky M. B. Addiction protein Phd of plasmid prophage P1 is a substrate of the ClpXP serine protease of Escherichia coli. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3274–3277. doi: 10.1073/pnas.92.8.3274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ludtke D. N., Austin S. J. The plasmid-maintenance functions of the P7 prophage. Plasmid. 1987 Jul;18(1):93–98. doi: 10.1016/0147-619x(87)90083-7. [DOI] [PubMed] [Google Scholar]
  30. Meacock P. A., Cohen S. N. Partitioning of bacterial plasmids during cell division: a cis-acting locus that accomplishes stable plasmid inheritance. Cell. 1980 Jun;20(2):529–542. doi: 10.1016/0092-8674(80)90639-x. [DOI] [PubMed] [Google Scholar]
  31. Miki T., Chang Z. T., Horiuchi T. Control of cell division by sex factor F in Escherichia coli. II. Identification of genes for inhibitor protein and trigger protein on the 42.84-43.6 F segment. J Mol Biol. 1984 Apr 25;174(4):627–646. doi: 10.1016/0022-2836(84)90087-1. [DOI] [PubMed] [Google Scholar]
  32. Miki T., Yoshioka K., Horiuchi T. Control of cell division by sex factor F in Escherichia coli. I. The 42.84-43.6 F segment couples cell division of the host bacteria with replication of plasmid DNA. J Mol Biol. 1984 Apr 25;174(4):605–625. doi: 10.1016/0022-2836(84)90086-x. [DOI] [PubMed] [Google Scholar]
  33. Mori H., Kondo A., Ohshima A., Ogura T., Hiraga S. Structure and function of the F plasmid genes essential for partitioning. J Mol Biol. 1986 Nov 5;192(1):1–15. doi: 10.1016/0022-2836(86)90459-6. [DOI] [PubMed] [Google Scholar]
  34. Ogura T., Hiraga S. Mini-F plasmid genes that couple host cell division to plasmid proliferation. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4784–4788. doi: 10.1073/pnas.80.15.4784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pansegrau W., Lanka E., Barth P. T., Figurski D. H., Guiney D. G., Haas D., Helinski D. R., Schwab H., Stanisich V. A., Thomas C. M. Complete nucleotide sequence of Birmingham IncP alpha plasmids. Compilation and comparative analysis. J Mol Biol. 1994 Jun 24;239(5):623–663. doi: 10.1006/jmbi.1994.1404. [DOI] [PubMed] [Google Scholar]
  36. Pattus F., Massotte D., Wilmsen H. U., Lakey J., Tsernoglou D., Tucker A., Parker M. W. Colicins: prokaryotic killer-pores. Experientia. 1990 Feb 15;46(2):180–192. [PubMed] [Google Scholar]
  37. Projan S. J., Carleton S., Novick R. P. Determination of plasmid copy number by fluorescence densitometry. Plasmid. 1983 Mar;9(2):182–190. doi: 10.1016/0147-619x(83)90019-7. [DOI] [PubMed] [Google Scholar]
  38. Roberts R. C., Burioni R., Helinski D. R. Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2. J Bacteriol. 1990 Nov;172(11):6204–6216. doi: 10.1128/jb.172.11.6204-6216.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Roberts R. C., Helinski D. R. Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2. J Bacteriol. 1992 Dec;174(24):8119–8132. doi: 10.1128/jb.174.24.8119-8132.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Roberts R. C., Ström A. R., Helinski D. R. The parDE operon of the broad-host-range plasmid RK2 specifies growth inhibition associated with plasmid loss. J Mol Biol. 1994 Mar 18;237(1):35–51. doi: 10.1006/jmbi.1994.1207. [DOI] [PubMed] [Google Scholar]
  41. Salmon M. A., Van Melderen L., Bernard P., Couturier M. The antidote and autoregulatory functions of the F plasmid CcdA protein: a genetic and biochemical survey. Mol Gen Genet. 1994 Sep 1;244(5):530–538. doi: 10.1007/BF00583904. [DOI] [PubMed] [Google Scholar]
  42. Schmidhauser T. J., Helinski D. R. Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria. J Bacteriol. 1985 Oct;164(1):446–455. doi: 10.1128/jb.164.1.446-455.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sia E. A., Roberts R. C., Easter C., Helinski D. R., Figurski D. H. Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2. J Bacteriol. 1995 May;177(10):2789–2797. doi: 10.1128/jb.177.10.2789-2797.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stalker D. M., Thomas C. M., Helinski D. R. Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2. Mol Gen Genet. 1981;181(1):8–12. doi: 10.1007/BF00338997. [DOI] [PubMed] [Google Scholar]
  45. Sternberg N., Hamilton D. Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. J Mol Biol. 1981 Aug 25;150(4):467–486. doi: 10.1016/0022-2836(81)90375-2. [DOI] [PubMed] [Google Scholar]
  46. Tabuchi A., Min Y. N., Kim C. K., Fan Y. L., Womble D. D., Rownd R. H. Genetic organization and nucleotide sequence of the stability locus of IncFII plasmid NR1. J Mol Biol. 1988 Aug 5;202(3):511–525. doi: 10.1016/0022-2836(88)90282-3. [DOI] [PubMed] [Google Scholar]
  47. Tabuchi A., Min Y. N., Womble D. D., Rownd R. H. Autoregulation of the stability operon of IncFII plasmid NR1. J Bacteriol. 1992 Dec;174(23):7629–7634. doi: 10.1128/jb.174.23.7629-7634.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Thomas C. M. Recent studies on the control of plasmid replication. Biochim Biophys Acta. 1988 Mar 31;949(3):253–263. doi: 10.1016/0167-4781(88)90150-9. [DOI] [PubMed] [Google Scholar]
  49. Tsuchimoto S., Ohtsubo E. Effect of the pem system on stable maintenance of plasmid R100 in various Escherichia coli hosts. Mol Gen Genet. 1989 Feb;215(3):463–468. doi: 10.1007/BF00427044. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES